US6191852B1 - Optical measurement system for detecting luminescence or fluorescence signals - Google Patents

Optical measurement system for detecting luminescence or fluorescence signals Download PDF

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Publication number
US6191852B1
US6191852B1 US09/170,482 US17048298A US6191852B1 US 6191852 B1 US6191852 B1 US 6191852B1 US 17048298 A US17048298 A US 17048298A US 6191852 B1 US6191852 B1 US 6191852B1
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Prior art keywords
measurement system
plate
image
glass
optical
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Wolfgang Paffhausen
Martin Bechem
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Bayer Intellectual Property GmbH
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Bayer AG
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6484—Optical fibres
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/028—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having reaction cells in the form of microtitration plates

Definitions

  • the invention is based on a measurement system for detecting optical signals of microassays, in which the signal-generating test objects are arranged on an investigation surface of a planar carrier, comprising an optical imaging arrangements which reduces the size of the test objects to be measured in such a way, that all the objects are imaged completely on a two-dimensional, photosensitive image sensor.
  • the optical signals are converted by the image sensor into electronic image signals, which are evaluated by a measurement computer in a known manner and processed further.
  • Test objects in the context of the invention are to be understood to mean fluorescent or luminescent and/or fluorescent- or luminescent-marked samples which are provided on the carrier or in microtitre plates and in which a chemoluminescent or bioluminescent reaction proceeds in the case of luminescence on account of molecular interactions, in which reaction photons can be liberated and detected, or fluorescence arises in the case of fluorescence on account of the interaction of a fluorescent die with which the objects are marked, given irradiation by suitable excitation energy, with the result that photons can be liberated and detected.
  • the samples themselves may be present in the form of dissolved chemical components or else in the form of biological test systems, such as for instance, in the case of enzymatic reactions, antigen-antibody couplings, protein binding assays, ligands-receptor interactions or receptor assays.
  • the biological test system may be configured as a cellular assay (adherent or suspension cells, primarily mammalian cells, but also plant cells, bacteria, fungi, yeasts or viruses) or else may comprise subcellular constituents, such as e.g. isolated cell nuclei or cytoplasm agglomerates, or else may comprise artificial carriers, such as e.g. plastic beads or glass microspheres, on which biologically active material, generally cellular or subcellular constituents, has been applied, an optical signal in the form of photons being liberated as a result of the interaction of different components.
  • a cellular assay adheredherent or suspension cells, primarily mammalian cells, but also plant cells, bacteria, fungi, yeasts or viruses
  • microtitre plates having dimensions of approximately 130 mm ⁇ 86 mm, depth approximately 10-14 mm, which contain 96, 384 or 1536 test holes, to be optically measured simultaneously using imaging methods
  • MTP microtitre plates
  • two-dimensional luminescence measurement systems are necessary for this.
  • the multiplied electrons impinge on a luminescent phosphor, where they engender, in a spatially resolved manner, a light signal which is amplified by up to 1,000,000 times relative to the input and can be detected, in a spatially resolved manner, using a CCD sensor.
  • An output image that has been intensified in such a way, can be evaluated with the aid of image-processing processes, the brightness in each hole of a microtitre plate being calculated as the number of recorded photon events.
  • Corresponding systems are commercially available as so-called MTP readers from various companies. If the intensities are sufficiently high and the integration time is unimportant, it is also possible to have recourse to commercially available cooled CCD systems instead of the image intensifier.
  • the microtitre plate in the case described here onto the photocathode of an image intensifier, all the manufacturers use an objective of high light-gathering power. Some manufacturers use standard photo objectives, others use specially corrected objectives having a high f-number. The best high-performance objectives used to date already have a very high light intensity with an aperture ratio of approximately 1:1.0 and a focal length of 50 mm. An optical arrangement having a significantly higher light-gathering power cannot be constructed for physical reasons.
  • the object In order to increase e.g. the capacity of a robot installation for the investigation of luminescence or fluorescence signals in microtitre plates by reducing the integration time for the measurement of microtitre plates and/or to reduce e.g. the number of cells per test hole and/or to reduce the size of expensive substrate quantities for an enzyme reaction, the object consisted in increasing the sensitivity of the known luminescence or fluorescence measurement systems.
  • the optical imaging arrangement comprises a high-resolution glass-fibre taper element having a large-area and a small-area end and the end surfaces are chosen such that the large-area end surface corresponds at least to the investigation surface of the carrier and the small-area end surface corresponds to the size of the image sensor, the ratio of the end surfaces producing the scale of reduction of the optical imaging arrangement in order to image the investigation surface of the carrier completely onto the image sensor.
  • “High-resolution” is in this case to be understood to mean that the fibre diameter of the densely packed glass fibres situated next to one another at the large end surface of the glass-fibre taper element is ⁇ 12 ⁇ m.
  • the invention is preferably realised with the aid of an arrangement in which the planar carrier comprises a microtitre plate having a multiplicity of holes for receiving the signal-generating test objects, the image sensor comprises an image intensifier and a video camera for converting the intensified image signals into electronic signals, and the glass-fibre taper element is designed in such a way that a reduced image of the microtitre plate that fully fills the entry window of the image intensifier is produced.
  • an image intensifier having a bialkali photocathode whose spectral sensitivity at wavelengths>700 nm is ⁇ 1% of its maximum sensitivity.
  • the microtitre plate is provided with a horizontally movable and vertically adjustable, drawer-like mount, which, after horizontal retraction, is raised to an extent such that the large-area end of the glass-fibre taper element is in direct contact with the microtitre plate.
  • the small-area end of the glass-fibre taper element is advantageously in direct optical contact with the entry window of the image intensifier.
  • an air gap remaining between the glass-fibre taper element and the entry window of the image intensifier is filled with an oil film, whose refractive index corresponds to the refractive index of the taper element.
  • the apparatus according to the invention is modified in such a way that the microtitre plate has an optically transparent base, in that the drawer-like mount is designed as a frame construction and in that a light source for fluorescence excitation which generates a light pencil of inclined incidence with respect to the optical axis is arranged underneath the microtitre plate.
  • a further development of the invention is characterized in that the entire apparatus can be pivoted through 180° about a horizontal spindle and has the following further features:
  • the microtitre plate has an optically transparent base.
  • the drawer-like slide mount for the microtitre plate is designed as a frame construction.
  • the measurement system additionally has a micropipetting system whose individual pipettes are assigned to the test holes in the microtitre plate.
  • An arrangement of this type permits in-situ observation of the luminescence of biological objects under the influence of an added reagent. This enables kinetic bioluminescence investigations which reproduce the dynamic action profile of pharmacologically active substances.
  • the new system is a factor of 10 more light sensitive than conventional microtitre plate luminescence measurement systems. In this way, very low-intensity bioluminescent reactions can actually be detected for the first time.
  • the integration time can be shortened by the factor of 10.
  • the quantity of biological objects e.g. the cell number/microtitre plates
  • the sample throughput can be increased by the factor of 10.
  • the substrate quantity which liberates the photons to be detected in a biochemical reaction can be reduced by the factor of 10.
  • FIG. 1 shows the fundamental structure of the optical measurement system
  • FIG. 2 a shows a diagrammatic view of a luminescence measurement system in the basic position (TOP position)
  • FIG. 2 b shows a diagrammatic view of the luminescence measurement system in a position (BOTTOM position) rotated through 180° relative to FIG. 2 a
  • FIG. 3 shows the structure of the measurement system for luminescent biological objects
  • FIG. 4 shows a plan view of a microtitre plate
  • FIG. 5 shows the drawer-like mount of the microtitre plate
  • FIG. 6 shows the fundamental structure of a measurement system for investigating fluorescent biological objects with excitation light of inclined incidence
  • FIG. 7 shows the fundamental structure of a luminescence measurement system in the BOTTOM position with a pipetting device for investigating dynamic processes in luminescent biological objects.
  • the invention utilizes the property of optical waveguides (glass or polymer fibres) of transmitting light signals, in particular including individual photons, in a wide spectral range.
  • An ordered arrangement of a large number of fibres in an xy area makes it possible to display a brightness image as intensity raster graphic at another location (image conductor).
  • image conductor For this purpose, each point xgi, ygi in the object plane g must correspond to a point xbi, ybi in the image plane b. If each fibre of the image conductor is geometrically tapered on its path from the object plane to the image plane by its diameter being reduced, then the object image is reduced by the
  • optical taper arrangement or taper element.
  • Such elements are manufactured industrially as optical image transmission elements.
  • optical image transmission elements there are two manufacturing companies able to process large-area taper elements, that is to say glass-fibre bundles up to a diameter of 147 mm, and produce them in accordance with customer specifications with regard to the desired scale of reduction.
  • An important optical imaging property is that both on the object side (large end surface of the taper element) and on the image side (small end surface of the taper element), the individual glass fibres are parallel to the optical axis and are thus directed perpendicularly into or onto the signal-generating test objects, thereby avoiding the disturbing vignetting effects (parallax errors) occurring in the case of lens imaging.
  • the input windows of the image intensifier photocathodes of commercial photon counting systems have a diameter of 12 mm, 18 mm, 25 mm or 40 mm. As the area becomes larger, the price of the image intensifiers increases more than proportionally. On the other hand, a minimum reduction factor fv is to be sought for the taper element because its aperture and hence its performance in the transmission of optical signals decreases at 1/fv. In the case of the present invention, an image intensifier with a diameter of 25 mm was selected as an acceptable compromise between costs and usefulness. This results in a computational numerical aperture of 0.167 in accordance with an aperture angle of approximately 20 degrees in comparison with the much smaller aperture angle of an f 1:1.0 objective at a distance of 70 cm of 0.1 degree.
  • the photon collecting property is improved with the aid of the taper element by a multiple relative to the conventional optical arrangement.
  • the sensitivity of a system for the transmission of optical signals was able to be increased by the factor of 10 with the aid of the optical imaging arrangement comprising the glass-fibre taper element.
  • the fundamental structure of the optical measurement system is evident in FIG. 1 .
  • the most important component in this case is the glass-fibre taper element 1 having a large-area end 2 and a small-area end 3 .
  • a planar carrier 4 Situated opposite the large end surface 2 is a planar carrier 4 with the test objects 5 arranged thereon, and situated opposite the small end surface 3 is an image sensor 6 for the acquisition and further processing of the reduced image of the carrier surface with the test objects 5 .
  • the taper element 1 has approximately a bell-shaped contour.
  • the individual glass fibres 7 are oriented parallel to the optical axis 8 and thus perpendicular to the respective surfaces 2 and 3 .
  • FIGS. 2 a and 2 b A pivotable luminescence measurement system is illustrated according to FIGS. 2 a and 2 b.
  • the carrier 4 is a microtitre plate with test holes 9 for the objects 5 to be investigated.
  • the measurement system essentially comprises the microtitre plate 4 , the glass-fibre taper element 1 , an image intensifier 10 and a CCD camera 11 .
  • the entire system is fitted on a fixed frame 13 in a manner allowing it to pivot about a horizontal rotary spindle 12 .
  • the taper element 1 is accommodated in a light-tight housing 14 .
  • the position according to FIG. 2 a corresponds to the basic position, in which the microtitre plate 4 is arranged as the lowest component and the optical elements 1 , 10 , 11 are arranged above it.
  • This position corresponds to the so-called TOP measurement position.
  • the entire system is pivoted through 180° about the rotary spindle 12 in the position according to FIG. 2 b.
  • the microtitre plate 4 is situated right at the top and, consequently, is also accessible from the top, while the optical components 1 , 10 , 11 are situated below the microtitre plate 4 .
  • This position corresponds to the so-called BOTTOM measurement position.
  • the light-tight housing 14 is rotated with the taper element 1 as optical reduction arrangement and the flanged-on photo detection system 10 , 11 as rigid unit.
  • the large-area end of the taper element 1 faces the microtitre plate 4 and its small-area end faces the image intensifier 10 .
  • the reduction factor of the taper element 1 is selected such that the reduced image of the microtitre plate 4 fully fills the entry window of the image intensifier 10 .
  • a reduction factor in the range from 1:2 to 1:6 is to be regarded as optimum in the case of measurement systems of this type.
  • FIG. 3 The actual structure of the optical system is evident in FIG. 3, in which the TOP position is again illustrated.
  • the glass-fibre entry window 15 on the image intensifier 10 it is necessary for the glass-fibre entry window 15 on the image intensifier 10 to be brought into contact with the small-area end 3 of the taper element 1 in order to ensure that the photons on the taper output side are efficiently transferred to the photocathode 16 of the image intensifier 10 .
  • an image intensifier having a bialkali photocathode is expediently resorted to, because the specific spectral sensitivity of such a photocathode prevents the negative property (associated with all white microtitre plates) of light-induced long-term photophorescence, which principally occurs at approximately 800 mm, from bringing about an unidentifiable false light signal and hence sensitivity limitation. Since the spectral sensitivity of the bialkali photocathode is virtually zero above 700 nm, the false light originating from the long-term phosphorescence is suppressed.
  • the gap 17 between taper element 1 and the input window 15 can be brought to a distance of zero in the z direction by means of adjusting screws 18 on the camera carrier plate 19 .
  • a spreading drop of oil 20 introduced into the gap 17 minimizes the reflection losses by matching the refractive index during the transition from the taper element 1 to the detection system 10 , 11 .
  • the oil has the same refractive index as the glass of the taper element 1 .
  • a rigid connection between the taper element 1 and the image intensifier 10 is established by the locking screws 21 , which connection is light-tight by means of a light trap 22 .
  • the microtitre plate 4 (also see FIG. 4) has a multiplicity of cylindrical or rectangular test holes 9 (so-called wells) for receiving the luminescent biological objects 5 .
  • the base 23 of the microtitre plate 4 is composed of an optically transparent material.
  • the mount in the form of a drawer for the microtitre plate 4 is explained in more detail with reference to FIG. 5 .
  • the measurement apparatus is loaded with the microtitre plate 4 with the aid of an extendable drawer 24 , which is designed in such a way that it can receive the microtitre plates 4 in an open frame construction 25 at least for the BOTTOM measurement method, so that the actual measurement surface is not covered.
  • the extending of the drawer 24 is coupled with an interruption of the high voltage of the image intensifier 10 , in order to protect the latter against destruction by too much light.
  • the microtitre plate surface 26 and the taper surface 2 have to be brought into contact. This is done by means of a resilient vertical adjustment 29 (only indicated diagrammatically in FIGS. 3 and 5 ), by means of which the microtitre plate 4 is raised against the taper element 1 after the microtitre plate 4 has been positioned precisely under the large end surface 2 of the taper element 1 , which surface yields, however, after a defined pressure force is exceeded. This enables microtitre plates 4 with different height dimensions to be adapted to the taper surface 2 without any play.
  • the base of the housing part 30 enclosing the drawer 24 contains a perforation which can be closed off by a dummy plate 31 (see FIG. 3 ).
  • the dummy plate is removed and replaced by a window 32 .
  • Fluorescent excitation light 33 can be radiated in through this window at an inclined angle of incidence in the TOP measurement position in order to be able to investigate fluorescent objects.
  • the base 23 of the microtitre plate 4 is likewise optically transparent.
  • An exchangeable, large-area interference filter 34 which is selective for the fluorescent light, is arranged between the large end surface 2 of the taper element 1 and the microtitre plate 4 in order to suppress interfering radiation from the excitation light 33 . Consequently, the apparatus described can be converted quickly and without difficulty from a luminescence measurement system to a fluorescence measurement system having the same high sensitivity.
  • FIG. 7 A further possibility for expansion of the luminescence measurement system is illustrated in FIG. 7 .
  • the apparatus which is in this case pivoted into the BOTTOM measurement position, is additionally equipped with a micropipetting system 35 , which is arranged above the microtitre plate 4 .
  • the individual pipettes 36 are assigned to the individual test holes 9 (wells) of the microtitre plate 4 .
  • the drawer-like mount 24 for the microtitre plate 4 is provided with a frame 25 (see FIG. 5) in order that the top side of the microtitre plate 4 is accessible for the micropipetting system 35 and the underside is accessible for observation of the luminescence.
  • the base 23 of the microtitre plate 4 is once again composed of an optically transparent material in order to observe the luminescent radiation of the samples 5 from the underside through the base 23 .
  • This expansion permits dynamic investigation of luminescent biological objects, e.g. under the influence of reaction liquids which can be fed in via the pipettes 36 .
  • Kinetic bioluminescence investigations having a temporal resolution of 40 ms (video standard) which reflect the dynamic action profile of pharmacologically active substances on the biological objects are possible in this way.
  • substance effects can be investigated only in this way.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Plasma & Fusion (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Photometry And Measurement Of Optical Pulse Characteristics (AREA)
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  • Spectrometry And Color Measurement (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
US09/170,482 1997-10-14 1998-10-13 Optical measurement system for detecting luminescence or fluorescence signals Expired - Lifetime US6191852B1 (en)

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DE19745373A DE19745373A1 (de) 1997-10-14 1997-10-14 Optisches Meßsystem zur Erfassung von Lumineszenz- oder Fluoreszenzsignalen
DE19745373 1997-10-14

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EP (1) EP0909947B1 (ja)
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DE (2) DE19745373A1 (ja)
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